Our proposal is designed to determine how intracellular pH (pHi) dynamics regulates epithelial plasticity, with a focus on distinct types of ell differentiation. Transitions in the fate or morphological state of epithelial cells are central to metazoan development, homeostasis, and tissue repair. Our preliminary data indicate that increased pHi is necessary for three types of epithelial differentiation programs, the transdifferentiation of epithelial to mesenchymal cells (EMT), adult epithelial stem cell differentiation, and embryonic stem (ES) cell differentiation. EMT is necessary for normal development, contributes to organ repair after injury, including aberrant repair leading to fibrosis, and promotes cancer metastasis. The genetically distinct process of adult stem cell self-renewal and differentiation is a fundamental part of the program of adult homeostasis and tissue repair. The differentiation of ES cells mimics the process of lineage specification and expansion during embryonic development. Hence, resolving how these three types of epithelial differentiation are regulated has broad significance for both normal and pathological cell behaviors. Our data support testing the central hypothesis that increased pHi is necessary for different types of epithelial cell differentiation. In Aim 1 we will identify molecular mechanisms or pHi-regulated EMT based on our findings that increased pHi is necessary for EMT of lung and mammary epithelial cells. We will reveal stage-specific pHi dynamics and its regulation of actin filament remodeling and transcriptional events during EMT by using real-time imaging of genetically encoded biosensors with clonal cell models in 2D and 3D cultures as well as in vivo analysis of zebrafish neural crest development. We also will identify molecular mechanisms mediating pHi-dependent EMT by testing established and predicted pH-sensing proteins identified using an analytical combination of protein structures, cancer mutation databases, and a newly developed bioinformatics program that identifies titrating ionizable residues in proteins. In Aim 2 we will determine how pHi regulates differentiation of adult and embryonic stem cell lineages based on our findings that increased pHi is necessary for in vivo differentiation of the Drosophila ovarian follicle stem cell lineage, and for spontaneous differentiation of mouse ES cells. We will resolve how pHi dynamics regulates established cell lineage markers and the role that pH sensors play in Drosophila follicle cell and mouse ES cell differentiation, including investigating pHi-dependent hedgehog and wingless signaling, as suggested by our preliminary data. We bring to these studies new views on signaling mechanisms at the molecular level from our expertise in bridging structural and cell biology, quantitative live cell imaging, and Drosophia genetics. Although pHi is routinely measured in tissue culture cells, few studies have investigated pHi dynamics in vivo. Successful completion of these studies will provide substantial insight into a significant and unstudied mechanism for the regulation of epithelial plasticity, revealing new regulators for therapeutic targeting of disease-associated differentiatio programs.